The goal of this project is to develop and evaluate methods for manufacturing dendritic cells (DCs) for clinical immunotherapy trials. In FY 1999, we developed and optimized a full-scale GMP method for 5-day flask culture of autologous DCs in RPMI, autologous plasma or allogeneic serum, IL4 and GMCSF, starting with peripheral blood monocytes collected by apheresis and purified by elutriation. The immature DCs generated are then available for further manipulations (e.g., peptide pulsing) prior to clinical administration. This manufacturing method was incorporated into several clinical trials, and a manuscript describing this method was published in early 2001. Because of our interest in developing closed systems and eliminating reagents that are difficult to standardize, we evaluated a 7-day culture system in a protein-defined, serum-free medium (XVIVO15) starting with monocytes from elutriation vs. negative immunomagnetic selection using the Isolex 300I, in bags vs. flasks. We demonstrated that the 2 different isolation methods for monocytes product equivalent immature DC populations, and that bags were equivalent to flasks. Furthermore, historical comparison showed that serum-free medium was equivalent and perhaps even superior to serum-containing medium for generation of immature DCs. A manuscript describing this work has been accepted for publication. Studies over the past year focused on evaluating culture conditions for generating mature DCs using CD40 ligand after culture in IL4 and GMCSF. A process was successfully developed and incorporated into several cancer immunotherapy trials in early 2001. Studies over the next year will focus on characterizing these DCs by flow cytometric phenotyping and on evaluating additional aspects of the manufacturing process, including stability of the source material (apheresis MNCs) and of the DC product after peptide pulsing.